Fluorescent-labelled bleomycin analogues

Abstract

A fluorescent-labelled bleomycin analog useful as a probe for measuring cellular uptake of bleomycin and bleomycin derivatives, including methods of making and using same. The analog may further be used as a therapeutic agent as well as a diagnostic tool.

We claim: 
     
       1. A fluorescent probe for detecting the intracellular uptake and distribution of a talisomycin, said probe being a fluorescent-labelled analogue which is a covalently linked product of said talisomycin having the structure ##STR4## where R═NH--(CH 2 ) 3  --CH(NH 2 )--CH 2  --CO--NH--(CH 2 ) 3  --NH(CH 2 ) 4  --NH 2  ; NH--(CH 2 ) 3  --NH--(CH 2 ) 4  NH 2  ; or NH--(CH 2 ) 4  --NH 2  and a fluorescent moiety, said fluorescent moiety being present in said product in a quantity of one molecule of said fluorescent moiety attached to said R group at the terminal amine of said talisomycin, said fluorescent-labelled analogue exhibiting a fluorescence intensity about 300-400 times greater than said talisomycin, said probe being characterized by retaining substantially the same biological properties as said talisomycin and being adapted to ascertain the content, uptake and distribution of said talisomycin in living cells. 
     
     
       2. The fluorescent probe of claim 1 wherein said fluorescent moiety is selected from the group consisting of fluorescein, 4-halo-7-nitrobenzo-2-oxa-1,3, diazole (NBD), 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene-3-propionic acid (BODIPY), pyrene, tetramethylrhodamine and cyanine dyes. 
     
     
       3. A fluorescent probe for detecting the intracellular uptake and distribution of a bleomycin, said probe being a fluorescent-labelled analogue which is a convalently linked product of said bleomycin having the structure ##STR5## where R═OH; NH--(CH2) 3  --S--CH 3  ; NH--(CH 2 ) 3  --NH 2  ; or NH--(CH 2 ) 3  --NH--(CH 2 ) 4  --NH 2  and a fluorescent moiety, said fluorescent moiety being present in said product in a quantity of one molecule of said fluorescent moiety attached to said R group at the terminal amine of said bleomycin, said fluorescent-labelled analogue exhibiting a fluorescence intensity about 300-400 times greater than said bleomycin, said probe being characterized by retaining substantially the same biological properties as said bleomycin and being adapted to ascertain the content, uptake and distribution of said bleomycin in living cells. 
     
     
       4. The fluorescent probe of claim 3 wherein said fluorescent moiety is selected from the group consisting of fluorescein, 4-halo-7-nitrobenzo-2-oxa-1,3, diazole (NBD), 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-S-indacene-3-propionic acid (BODIPY), pyrene, tetramethylrhodamine and cyanine dyes. 
     
     
       5. A fluorescent probe for detecting the intracellular uptake and distribution of a talisomycin, said probe being a fluorescent-labelled analogue which is a covalently linked product of said talisomycin having the structure ##STR6## where R═NH--(CH 2 ) 3  --CH(NH 2 )--CH 2  --CO--NH--(CH 2 ) 3  --NH(CH 2 ) 4  --NH 2  ; NH--(CH 2 ) 3  --NH--(CH 2 ) 4  NH 2  ; or NH--(CH 2 ) 4  --NH 2  and a fluorescent moiety, said fluorescent moiety being present in said product in a quantity of one molecule of said fluorescent moiety attached to said R group at the terminal amine of said talisomycin, said fluorescent-labelled analogue exhibiting a fluorescence intensity about 300-400 times greater than said talisomycin, said probe being characterized by retaining substantially the same biological properties as said talisomycin and being adapted to ascertain the content, uptake and distribution of said talisomycin in living cells, and wherein retention of said biological properties of talisomycin facilitates fragmentation of DNA in said living cells. 
     
     
       6. A fluorescent probe for detecting the intracellular uptake and distribution of a talisomycin, said probe being a fluorescent-labelled analog which is a covalently linked product of said talisomycin having the structure ##STR7## where R═NH--(CH 2 ) 3  --CH(NH 2 )--CH 2  --CO--NH--(CH 2 ) 3  --NH(CH 2 ) 4  --NH 2  ; NH--(CH 2 ) 3  --NH--(CH 2 ) 4  NH 2  ; or NH--(CH 2 ) 4  --NH 2  and a fluorscent moiety, said fluorescent moiety being present in said product in a quantity of one molecule of said fluorescent moiety attached to said R group at the terminal amine of said talisomycin, said fluorescent-labelled analogue exhibiting a fluorescence intensity about 300-400 times greater than said talisomycin, said probe being characterized by retaining substantially the same biological properties as said talisomycin and being adapted to ascertain the content, uptake and distribution of said talisomycin in living cells, and wherein retention of said biological properties of talisomycin facilitates wherein said fluorescent moiety is selected from the group inhibition of cell growth. 
     
     
       7. A fluorescent probe for detecting the intracellular uptake and distribution of a talisomycin, said probe being a fluorescent-labelled analogue which is a covalently linked product of said talisomycin having the structure ##STR8## where R═NH--(CH 2 ) 3  --CH(NH 2 )--CH 2  --CO--NH--(CH 2 ) 3  --NH(CH 2 ) 4  --NH 2  ; NH--(CH 2 ) 3  --NH--(CH 2 ) 4  NH 2  ; or NH--(CH 2 ) 4  --NH 2  and a fluorescent moiety, said fluorescent moiety being present in said product in a quantity of one molecule of said fluorescent moiety attached to said R group at the terminal amine of said talisomycin, said fluorescent-labelled analogue exhibiting a fluorescence intensity about 300-400 times greater than said talisomycin, said probe being characterized by retaining substantially the same biological properties as said talisomycin and being adapted to ascertain the content, uptake and distribution of said talisomycin in living cells, and wherein retention of said biological properties of talisomycin facilitates measurement of sensitivity and resistance to talisomycin in living cells. 
     
     
       8. A fluorescent probe for detecting the intracellular uptake and distribution of a bleomycin, said probe being a fluorescent-labelled analogue which is a covalently linked product of said bleomycin having the structure ##STR9## where R═OH; NH--(CH 2 ) 3  --S--CH 3  ; NH--(CH 2 ) 3  --NH 2  ; or NH--(CH 2 ) 3  --NH--(CH 2 ) 4  --NH 2  and a fluorescent moiety, said fluorescent moiety being present in said product in a quantity of one molecule of fluorescent moiety attached to said R group at the terminal amine of said bleomycin, the analogue exhibiting a fluorescence intensity about 300-400 times greater than said bleomycin, said probe being characterized by retaining substantially the same biological properties as said bleomycin and being adapted to ascertain the content, uptake and distribution of said bleomycin in living cells, and wherein retention of said biological properties of bleomycin facilitates fragmentation of DNA in said living cells. 
     
     
       9. A fluorescent probe for detecting the intracellular uptake and distribution of a bleomycin, said probe being a fluorescent-labelled analogue which is a covalently linked product of said bleomycin having the structure ##STR10## where R═OH; NH--(CH 2 ) 3  --S--CH 3  ; NH--(CH 2 ) 3  --NH--(CH 2 ) 4  --NH 2  and a fluorescent moiety, said fluorescent moiety being present in said product in a quantity of one molecule of fluorescent moiety attached to said R group at the terminal amine of said bleomycin, the analogue exhibiting a fluorescence intensity about 300-400 times greater than said bleomycin, said probe being characterized by retaining substantially the same biological properties as said bleomycin and being adapted to ascertain the content, uptake and distribution of said bleomycin in living cells, and wherein retention of said biological properties of bleomycin facilitates inhibition of cell growth. 
     
     
       10. A fluorescent probe for detecting the intracellular uptake and distribution of a bleomycin, said probe being a fluorescent-labelled analogue which is a covalently linked product of said bleomycin having the structure ##STR11## where R═OH; NH--(CH 2 ) 3  --S--CH 3  ; NH--(CH 2 ) 3  --NH 2  ; or NH--(CH 2 ) 3  --NH--(CH 2 ) 4  --NH 2  and a fluorescent moiety, said fluorescent moiety being present in said product in a quantity of one molecule of fluorescent moiety attached to said R group at the terminal amine of said bleomycin, the analogue exhibiting a fluorescence intensity about 300-400 times greater than said bleomycin, said probe being characterized by retaining substantially the same biological properties as said bleomycin and being adapted to ascertain the content, uptake and distribution of said bleomycin in living cells, and wherein retention of said biological properties of bleomycin facilitates measurement of sensitivity and resistance to bleomycin in living cells. 
     
     
       11. The fluorescent probe of claim 1 made by a process comprising: a. providing talisomycin;   b. preparing a metal complex of said talisomycin in solution;   c. buffering said metal complex containing solution with buffered means;   d. adding a buffered fluorescent moiety-containing solution to said buffered metal complex-containing solution;   e. mixing said solutions; and   f. recovering a fluorescent-labelled derivative of talisomycin.   
     
     
       12. The fluorescent probe of claim 11 wherein the metal in step b. is selected from the group consisting of Zn, Cu, Cd and Co and preparing said metal complex by adding a salt of said metal to an aqueous solution of said talisomycin. 
     
     
       13. The fluorescent probe of claim 3 made by a process comprising: a. providing bleomycin;   b. preparing a metal complex of said bleomycin in solution;   c. buffering said metal complex containing solution with buffered means;   d. adding a buffered fluorescent moiety-containing solution to said buffered metal complex-containing solution;   e. mixing said solutions; and   f. recovering a fluorescent-labelled derivative of bleomycin.   
     
     
       14. The fluorescent probe of claim 11 wherein the metal in step b. is selected from the group consisting of Zn, Cu, Cd and Co and preparing said metal complex by adding a salt of said metal to an aqueous solution of said bleomycin.